The present invention relates to a multi-epitope peptide, which is useful for peptide-based immunotherapy of allergic diseases.
Allergic diseases are defined to be functional disturbances caused by type I hypersensitivity (type I immune response mediated by IgE antibodies) or a kind of disease induced by the disturbance. The symptoms include pollinosis, bronchial asthma, allergic rhinitis, atopic dermatitis, and anaphylactic shock. Pollinosis is a representative allergic disease. In Japan, approximately 10% of the population suffers from cedar pollinosis, and the number of the patients is still increasing. In America, 5 to 15% of the population suffers from short ragweed pollinosis. Pollinosis is a serious problem both socially and economically because there are many patients and they suffer from unbearable conditions such as itchiness of eyes, runny noses, sneezing, and nasal congestion. Moreover, once the patient acquires pollinosis, the disease manifests itself every year. An effective therapy for pollinosis has thus earnestly been sought.
To comprehend and treat allergic diseases, it is important to understand how a type I allergic response is developed. Current studies focus on clarifying the initial reaction in the allergen-specific immune response, especially the mechanism of regulating a T cell-mediated allergic reaction. Initiation of an immune response to a foreign antigen including an allergen depends on antigen-presenting cells in the immune system. The antigen-presenting cells (i.e., B cells, macrophages, and dendritic cells) take up incoming foreign antigens, break them down to antigen peptides (T cell epitope peptides), put the fragments in a pocket consisting of xcex1 and xcex2 chains of major histocompatibility complex (MHC) class II molecules (HLA class II in human), display the fragments on the cell surface, and thereby present the foreign antigens to antigen-specific CD4 positive helper T cells (Th cells). An HLA class II molecule consists of DR, DQ and DP molecules. The xcex1-chain of the DR molecule is encoded by the HLA-DRA gene, and the xcex2-chain is encoded by the HLA-DRB1, -DRB3, -DRB4 or -DRB5 gene. The xcex1-chain of the DQ molecule is encoded by the HLA-DQA1 gene, and the, xcex2chain is encoded by the HLA-DQB1 gene. The xcex1-chain of the DP molecule is encoded by the HLA-DPA1 gene, and the xcex2-chain is encoded by the HLA-DPB1 gene. Each gene except for HLA-DRA contains many alleles. The pocket in which antigenic peptides are placed is highly polymorphic, and the structures differ slightly from each other. Because of this, the kind of antigenic peptides that bind to the pocket and are presented to T cells is restricted to that structure.
Once Th cells receive HLA class II-restricting antigen information via the T cell receptor (TCR), they are activated to secrete various cytokines, by which they proliferate by themselves. At the same time, the Th cells induce differentiation of B cells into plasma cells, to induce antibody production. Depending upon the difference in the cytokine-producing pattern, the Th cells activated by antigen stimulation are classified into Th 1 cells capable of producing interferon 2 (IL-2), interferonxcex3 (IFN-xcex3) and lymphotoxin (TNF-xcex2); Th 2 cells capable of producing IL-4, IL-5, IL-6, IL-10 and IL-13; and Th0 cells capable of producing both cytokines. The production of IgE antibody, which is a cause of allergy, is promoted by IL-4 and IL-13 but suppressed by IFN-xcex3. That is, Th1 cells suppress IgE production, whereas Th2 cells promote IgE production. In other words, sensitization of allergy is determined by whether Th1 cells or Th2 cells function upon exposure of antigens. It is commonly known that Th2 cells predominantly function in the patients with allergy. Allergen-specific IgE antibodies adhere to peripheral basophil and tissue mast cells. The subsequent exposure of allergen results in cross-linking of the IgE antibody on the basophil or the mast cell via the allergen. This releases inflammatory mediators including histamine, prostaglandins, and leucotriene, thereby causing an immediate allergy response. In response to these inflammatory mediators, lymphocytes, monocytes, basophils, and eosinophils are localized in the inflammatory region of the tissue and result in the release of mediators that cause various reactions including disturbance and a late phase reaction.
One way to treat a particular allergy by antigen-specifically suppressing IgE antibody production is hyposensitization therapy using an allergen protein molecule. Hyposensitization therapy can provide a long-term effect that cannot be achieved by chemotherapy, and hence, is the only treatment close to an effective therapy. However, hyposensitization therapy is not always accepted as a general method for treating allergy, possibly because its mechanism and possible side effects (such as topical swelling or anaphylactic shock) remain unknown.
In place of hyposensitization therapy, a mechanism of hyposensitization using a peptide antigen bearing a T cell epitope has been proposed. The peptide fragment carrying a T cell epitope on the allergen molecule used for this therapy contains no B cell epitope or, if any, is monovalent so that the peptide fails to cross-link an IgE receptor with high affinity on the mast cell. For these reasons, patients administered the peptide fragment should not experience side effects such as anaphylactic shock. It is further known that when T cell epitope is given in vivo, T cells are antigen-specifically inactivated (anergy) (La Salle J. M. et al.: J. Exp. Med. 176: 177-186, 1992). It is reported that based on such a theoretical background, hyposensitization using a peptide carrying major T cell epitopes of cat dander allergen Fel d1 was carried out in an experimental murine model, and T cell anergy was induced in vitro (Briner, T. J. et al.: Proc. Natl. Acad. Sci. USA, 90: 7608-7612, 1994). Clinical trials on hyposensitization using this peptide are now under way (Norman, P. S. et al.: Am. J. Respir. Crit. Care Med. 154: 1623-1628, 1996; Simons, F. E. et al.: Int. Immunol. 8: 1937-1945, 1996). Hyposensitization therapy using such a peptide carrying the major T cell epitope on the allergen molecule is called xe2x80x9cpeptide-based immunotherapyxe2x80x9d (or xe2x80x9cpeptide-based hyposensitization therapyxe2x80x9d).
As a standard for selecting T cell epitope peptides appropriate for the peptide-based immunotherapy, a positivity index (a mean T cell stimulation index multiplied by appearance frequency) is proposed in WO 94/01560. It is also reported that in peptide design, HLA haplotypic variations in a population of patients should be covered (Wallner, B. P. and Gefter M. L.: Allergy, 49: 302-308, 1994).
Generally, allergic patients have specific IgE antibodies to each of two or more allergen molecules differing from each other. For a potent allergy therapy, it is important to develop a peptide-based immunotherapeutic agent effective for these patients. However, such an immunotherapeutic agent has not yet been developed. Even the idea of such an agent has never been published in any of the above literatures. Accordingly, an objective of the present invention is to provide a peptide-based immunotherapeutic agent that is efficacious even for allergy patients sensitive to two or more different allergens.
Cedar pollen contains two major allergens, Cry j 1 (Yasueda, H. et al.: J. Allergy Clin. Immunol. 71: 77-36, 1983) and Cry j 2 (Taniai, M. et al.: FEBS Letter 239: 329-332, 1988; Sakaguchi, M. et al.: Allergy 45: 309-312, 1990). More than 90% of the patients with cedar pollinosis possess specific IgE antibodies to Cry j 1 and Cry j 2; the remaining patients (slightly less than 10%) possess a specific IgE antibody to either Cry j 1 or Cry j 2 (Hashimoto, M. et al.: Clin. Exp. Allergy 25: 848-852, 1995). Use of one or more T cell epitopes from only Cry j 1 or Cry j 2 would be expected to be less effective since IgE from the patients is reactive to both Cry j 1 and Cry j 2. Thus, T cell, eptopes from both Cry j 1 and Cry j 2 should be chosen to elevate the efficacy of the peptide-based immunotherapy for cedar pollinosis. Therefore, the present inventors prepared a multi-epitope peptide containing T cell epitopes of both Cry j 1 and Cry j 2 in the same molecule. They found that the multi-epitope peptide activated T cells of patients with pollinosis in vitro but did not react with IgE antibodies of the patients. They also found that an immune response was induced in vivo using mice. Based on these new findings, the inventors found that the multi-epitope peptide in this invention is effective as a peptide-based immunotherapeutic agent for patients with cedar pollinosis.
There are many cases of cedar pollinosis that also show clinical symptoms of Japanese cypress pollens. In view of this and based on the above invention, the present inventors prepared a multi-epitope peptide containing the T cell epitopes of Japanese cypress pollen allergen Cha o 1 (Japanese Patent Application No. Hei 8-153527) and the T cell epitopes of cedar pollen allergen Cry j 1 in the same molecule. The multi-epitope peptide activated T cells of both the patients with cedar pollinosis and the patients with Japanese cypress pollinosis, though these T cells do not react with each of the T cell epitopes. The multi-epitope peptides can thus be designed for T cell epitopes derived from not only cedar and Japanese cypress pollen allergens but also other various allergens.
HLA haplotype was investigated in a group of patients (including different races) as a criterion for selecting T cell epitopes to design multi-epitopes that are effective for a broader range of patients. T cell epitope peptides were selected noting that those binding to HLA whose haplotype frequently appears in the population and those presented on different HLA class II molecules, not the same HLA class II molecule, should be selected. The thus-selected multi-epitope peptides were clarified to be effective for a wider range of patients.
The present invention includes the inventions described in each claim.
The present invention will be described below in view of designing of multi-epitope peptides effective for the patients sensitive to cedar pollens or Japanese cypress pollens or the patients sensitive to both pollens, but this invention applies to patients sensitive to other allergens as well. The technical concept of the present invention also applies to plant pollens such as short ragweed (Amb a 1, Amb a 2, Amb a 5, Amb t 5, Amb p 5), Dactylis glomerata (Dac g 2), and Lolium perenne (Lol p 1, Lol p 2, Lol p 3); tree pollens such as Alnus glutinosa (Aln g 1), birch tree or Betula verrucosa (Bet v 1, Bet v 2), mountain cedar (Jun s 1), and juniper tree (Jun v 1); and various other allergens not specifically described herein.
The xe2x80x9cmulti-epitope peptidexe2x80x9d, used herein means a peptide molecule prepared by linearly joining peptides containing T cell epitopes derived from different allergen molecules (sometimes referred to as an antigenic peptide or merely as a peptide). In this peptide, a region that is cleaved in vivo is preferably inserted between the T cell epitope-containing peptides to minimize the occurrence of epitope sites that are newly recognized. The multi-epitope peptide is finally broken down to the respective antigenic peptides at the cleavage site. When administered, it can exhibit the effect comparable to that of a mixture of these respective antigenic peptides. The cleavage site may take any structure so long as it undergoes cleavage in vivo. Examples of the cleavage site include an arginine dimer and a lysine dimer that are recognition sequences of cathepsin B, which is an enzyme localized in lysosome.
Designing of the multi-epitope peptide according to the present invention will be described with reference to cedar pollen allergens Cry j 1 and Cry j 2 as examples.
Peripheral lymphocytes collected from the patients with cedar pollinosis are stimulated by Cry j 1 or Cry j 2 to produce the T cell line for individual patient. The T cell line is stimulated by an overlapping peptide consisting of about 15 amino acids, which covers the full-length primary structure of Cry j 1 (WO 94/01560) or Cry j 2 (Komiyama, N. et al.: Biochem. Biophys. Res. Commun. 201: 1201, 1994) to identify the antigenic peptides containing T cell epitopes in the Cry j 1 or Cry j 2 sequence (FIGS. 1 and 2).
Next, typing is performed for HLA class II molecules which bind to these antigenic peptides.
In humans, three different molecules, regions DR, DQ, and DP, exist as gene products of the HLA class II. This suggests that differentiation of T cells would be restricted by antigen-presenting molecules DR, DQ, and DP. The T cell clones established for each, patient are used to determine by which locus-derived antigen-presenting molecules the antigenic peptide of Cry j 1 or Cry j 2 is presented. They also determine whether the T cells that have received antigenic peptide information via DR, DQ or DP molecules tend to be differentiated into Th1 cells or Th2 cells. Such a typing is performed using the T cell clone established for individual patients (FIGS. 3 and 4).
FIGS. 3 and 4 clearly show that differentiation into Th1, Th2 or Th0 of the T cells stimulated by the antigenic peptide is not restricted by a specific epitope or a specific combination of HLA molecules. In selecting a peptide for designing the multi-epitope peptide of the present invention, any peptide can be a candidate for the antigenic peptide since any T cell epitope-containing peptide can stimulate T cells.
The criteria for selecting peptides to design the multi-epitope peptide of the present invention are as follows:
(1) Peptides are selected in the order of a positivity index (WO 94/01560) (peptides having a positivity index of 100 or more should be selected).
(2) Peptides presented on HLA class It molecules that frequently appear as antigen-presenting molecules are selected.
(3) Where there is no significant difference in the positivity index, peptides presented by restriction molecules of different types are selected to enhance the effectiveness. Specifically, in selecting a T cell epitope of an allergen that causes a certain allergic disease, the HLA haplotype in a group of patients with the allergy is first examined, and a T cell epitope restricted by an HLA haplotype whose gene frequency is high in the population to which the patient group belongs is selected. This is then the best selection that should achieve the best effect in that patient group. However, the thus-selected T cell epitope may not be effective at all in other patient groups.
Taking HLA haplotype DPB1*10501 as an example, it is assumed that this HLA haplotype is quite frequently observed in Japanese patients with a certain allergic disease, and the HLA haplotype-restricting T cell epitope is selected. The thus-selected peptide would hardly be effective for Northern American patients with the same allergy because the gene frequency of the HLA haplotype is as much as 39.0% in Japanese patients, whereas it is as little as 1.3% in white Americans and 0.8% in African Americans in Northern America. For Northern American patients, the HLA-DP restricting T cell epitope DPB1*0401 (in Northern America, 30.2% for white American patients and 11.1% for African American patients; 4.8% for Japanese patients) should be selected. It is also important to select a peptide presented on the antigen-presenting molecules differing in the locus level like DR, DQ, or DP; even though the loci are the same, it is important to select a peptide presented on the antigen-presenting molecules having different haplotypes.
In this case, the preferable epitope site contains no cysteine residue. When the epitope site contains a cysteine residue, the residue might bind non-specifically to HLA class II molecules. When immunized with an antigenic peptide containing a cysteine residue, the site that is originally not an antigen might be recognized as a new epitope. When such a peptide is recognized as an epitope, the cysteine-containing epitope is recognized by the peptide repeatedly through its second and third administrations, which may possibly cause side effects.
Specific embodiments of designing the multi-epitope will be described below. According to the positivity index of Cry j 1 and Cry j 2 shown in FIGS. 1 and 2, the T cell epitope of Cry j 1, Peptide No. 43 with amino acid residues at positions 211-225 (hereinafter abbreviated as p211-225) (restriction molecules DPA1*0101 to DPB1*0501) shows the highest positivity index and Peptide No. 22, p106-120 (restriction molecule DRB5*0101) shows the second highest positivity index. These two peptides are selected as the antigenic peptides to be used in the multi-epitope peptide. Turning to Cry j 2, Peptide No. 14, p66-80 (restriction molecule DRB5*0101) and Peptide No. 38, p186-190 (DRB4*0101) show the highest positivity indexes. Likewise, these two peptides can be selected as the antigenic peptides. Peptide No. 37, p181-195, located before Peptide No. 38 in Cry j 2 has a high positivity index of 280, but its restriction molecules are DPA1*0101 to DPB1*0201, which differ from the restriction molecule of Peptide No. 38. Since Peptide No. 37, p181-195 overlaps with Peptide No. 38, p186-200 by 10 residues, 5 residues from No. 37 are added ahead to No. 38. The thus-designed peptide can be selected as an HLA-DP restricting peptide. Peptides as selected above do not restrict DQ. Restriction molecules for Peptide No. 4, p16-30 of Cry j 1, are DQA1*0102 to DQB1*0602, but a cysteine residue is contained at the center of the epitope. Thus, Peptide No. 4 cannot be selected. In Cry j 2, p341-360, corresponding to Peptide Nos. 69-70, is a peptide presented on DQA1*0102 to DQB1*0602. Peptide No. 70 also contains cysteine, whereas T cells can be activated by only the cysteine-free Peptide No. 69. Thus, 12 residues, p344-355 (ISLKLTSGKIAS, SEQ ID NO. 6) be selected. Peptide No. 22, p106-120 of Cry j 1 contains cysteine at position 107. At least nine residues of p109-117 (FIKRVSNVI, SEQ ID NO. 7) are required for determining the T cell epitope core sequence using a T cell clone. Thus, if Pro-Cys residues at p106-107 are removed, the remaining peptide can be used.
The antigen taken up into the antigen-presenting cells is degraded in lysosome. How the foreign proteins taken up into the antigen-presenting molecules are processed and how they are bound to HLA class II molecules are still unknown. However, it is reported that cathepsin B participates in the digestion of antigens in this complicated mechanism (Katsunuma, N.: Nihon Men-Eki Gakkai (Japanese Society of Immunology) 25: 75, 1995).
With respect to several HLA class II types, an HLA-binding amino acid motif of the antigenic peptide has been determined. Binding to HLA class II molecules has specificity, but numerous antigenic peptides can bind to specific HLA class II molecules if the peptides meet a certain criterion (Rammensee, H. G. et al. Immunogenetics. 41: 178-228, 1995). For this reason, a newly recognized epitope site might possibly be created in the antigenic peptide-binding site. To avoid this, the multi-epitope peptide should be designed so as to be cleaved into each of the antigenic peptides in the antigen-presenting cells. The peptide sequence recognized by cathepsin B is the Arg-Arg-hydrophobic sequence or the Lys-Lys-hydrophobic sequence. Therefore, Arg-Arg or Lys-Lys is added to the latter half of the peptide containing the epitope and, in the following epitope sequence, a hydrophobic amino acid sequence is placed after Arg-Arg or Lys-Lys.
Since Arg-Arg is inserted between the antigenic peptides, the order of the antigenic peptides in this specific embodiment is considered insignificant. When Arg is linked to the latter half of Peptide No. 14 of Cry j 2 (FIG. 2), however, Tyr at position 73 becomes a first anchor. The Arg residue added then becomes amino acid residue at position 9 in the peptide motif of DRB5*0101 and serves as a second anchor. Thus, the Arg residue may be recognized as a new epitope. Therefore, this sequence should be located at the end of the multi-epitope peptide.
The thus-obtained multi-epitope peptide is shown as SEQ NO: 1. The restriction molecules for this multi-epitope are DRB4*0101, DRB5*0101, DPA1*0101-DPB1*0201, DPA1*0101-DPB1*0501, and DQA1*0102-DQB1*0602. In The 11th International Histocompatibility Workshop, the frequency of these genes was calculated in the Japanese population (Tsuji, K. et al.: HLA 1991, vol. 1, 1992, Oxford University Press) and found to be 0.291 for DRB4*0101, 0.056 for DRB5*0101 (0.070 for DRB5*0102), 0.208 for DPB1*0201, 0.399 for DPB1*0501, and 0.053 for DQB1*0602 (0.204 for DQB1*0601). Based on these data, the antigen frequency is calculated to be 0.50 for DRB4*0101, 0.11 for DRB5*0101 (0.14 for DRB5*0102), 0.37 for DPB1*0201, 0.64 for DPB1*0501, (0.79 according to Hori et al.), and 0.10for DQB1*0602 (0.37 for DQB1*0601). Since DRB5*0101 and DQB1*0602 are regarded as identical due to the presence of linkage disequilibrium, the data of DRB5*0101 is used for DQB1*0602. The probability that the Japanese population carries both DPB1*0201 and DPB1*0501 or either one is calculated to be 0.85. Similarly, the probability that the Japanese population carries both of DRB4*0101 and DRB5*0101 or either one is calculated to be 0.56. From these values, about 90% of patients are estimated to recognize more than one T cell epitope contained in the multi-epitope peptide of SEQ NO: 1. However, it is unclear whether the patients with these HLA types possess a T cell repertory capable of recognizing these epitope peptides presented on these restriction molecules. Furthermore, the number of epitopes that cause proliferation of T cells is unknown (two or more epitopes would be necessary). Thus, the efficiency of the multi-epitope peptide might decrease. In practice, it is properly assumed to be approximately 77% based on the result of testing proliferation response of peripheral lymphocytes from 17 patients.
To increase the range of patients to be effectively treated, the multi-epitope peptide can also be designed to carry more T cell epitopes than described above. Examples of such multi-epitope peptides include one prepared by joining p213-225 and p108-120 of Cry j 1, p182-200 and p79-98 of Cry j 2, p80-95 of Cry j 1, and p66-80 of Cry j 1, in this order (SEQ NO: 2), and one prepared by joining p213-225 and p108-120 of Cry j 1, p182-200 and p79-98 of cry j 2, p67-95 of Cry j 1, and p238-251 and p66-80 of Cry j 2, in this order (SEQ NO: 3). These multi-epitope peptides are effective as peptide-based immunotherapeutic agents since the peptides stimulated all the peripheral lymphocyte samples from the 21 tested patients with cedar pollinosis but did not react with the IgE antibody of the patients. Developing this concept, the effectiveness can be improved by preparing a T cell epitope containing allergens of different species, e.g., both Japanese cypress pollen allergen and cedar pollen allergen, by the method described in Example 13.
The present invention also includes modification of the antigenic peptide region used in the multi-epitope peptide to regulate the activity of T cells. The xe2x80x9cmodificationxe2x80x9d used herein means substitution, deletion, and insertion of at least one amino acid residue. Changes of properties of T cells imparted by amino acid substitution in the antigenic peptide can be examined by known methods. For example, 1) a certain amino acid of the multi-epitope peptide of the present invention is substituted with an analogous amino acid, e.g., by substituting Asp with Glu, Asn with Gln, Lys with Arg, Phe with Tyr, Ile with Leu, Gly with Ala, and Thr with Ser, to produce analog peptides, which are compared with the original peptide in T cell proliferating ability or lymphokine-producing ability. Alternatively, 2) a certain amino acid of the multi-epitope peptide is substituted with a non-analogous amino acid, for example, by substituting a polar amino acid or a hydrophilic amino acid with a hydrophobic amino acid Ala, and a hydrophobic amino acid with a hydrophilic amino acid Ser, and the property of the modified peptide is compared to that of the original peptide. The present invention also includes the thus-prepared multi-epitope analog peptides that are immunologically equivalent to the multi-epitope peptide of the present invention in terms of the positivity index and the T cell activation ability.
Most T cells that react with the antigenic peptide derived from Cry j 1 or Cry j 2 possess the properties of Th2 and Th0 in combination (FIGS. 3 and 4) BCG vaccine can potentiate the cellular immune activity to prevent infection with tubercle bacillus. To potentiate cellular immunity, T cells of Th1 type should be induced. It is reported that studies on the property of a human T cell clone with BCG inoculation revealed an increased level of Th1 type T cells (Matsushita, Sho, The 45th Japanese Association of Allergy, 836, 1995). According to Matsushita, there is a Th1 clone that is restricted by HLA-DR14 (DRB1*140) and that recognizes 84-100 amino acid sequence (EEYLILSARDVLAVVSK, SEQ ID NO. 8) of BCGA protein. If the HLA haplotype DPA1-DPB1*0501-restricting T cell epitope that is possessed by more than 60% of Japanese population is selected (for example, Peptide No. 43 (p211-225)/KSMKVTVAFNQFGPN (SEQ ID NO. 9) of Cry j 1 shown in FIG. 1), this peptide is bound to the 84-100 T cell epitope of tubercle bacillus BCGa protein restricted by DRB1*1405. It is highly likely that the thus-prepared multi-epitope peptide EEYLILSARDVLAVVSKRRMKVTVAFNQFGPN (SEQ ID NO. 10) would be quite efficacious for patients with cedar pollinosis carrying haplotype DRB1*1405. The use of such a multi-epitope peptide would lead to production of Th1 lymphokines, especially IL-12, by a peptide derived from BCGA antigen. It is known in several cases in humans and mice that IL-12 has an activity contradictory to that of IL-4 and acts on T cells to induce differentiation of Th cells to Th1 (Manetti, R., et al.: J. Exp. Med., 177, 1199-1204, 1993; Wu, C., et al.: J. Immunol., 151, 1938-1949, 1993; Hsieh, C. , et al.: Science, 260, 547-549, 1993). In particular, the experimental results by Manetti et al. indicate that a T cell clone specific to Der p1 antigen, a mite allergen, basically induces Th2 but induces Th1 or Th0 in the presence of IL-12. Thus, using the multi-epitope peptide prepared by joining a T cell epitope having Th1 induction activity to an allergen-reactive T cell epitope, T cells that are inherently induced to Th2 would be induced to Th1 or Th0.
When the peptide of the present invention containing at least one T cell epitope of Cry j 1 and/or Cry j 2 is subcutaneously administered to a mouse, which is then exposed to cedar pollen allergen, T cell anergy occurs (FIGS. 13 and 14), and IL-2 production is significantly reduced as compared to the control group. It is reported that hyposensitization therapy reduces IL-2 in humans (J. Allergy Clin. Immunol. 76: 188, 1985). Furthermore, the multi-epitope peptide of the present invention can activate each of the peptide-constituting T cell clones to the T cell epitope peptides (FIG. 10) but does not react with IgE antibodies of the patients (FIG. 8). These results show that the multi-epitope peptide of the present invention induces immune tolerance against allergens and is effective as a peptide-based immunotherapeutic agent for allergic diseases. The multi-epitope peptide of the present invention may be administered together with pharmaceutically acceptable carriers or diluents. The effective dose of the multi-epitope peptide may vary depending upon sensitivity to cedar pollen allergen, age, sex, and the body weight of the patients and other factors such as ability of a peptide to induce immune response in the patients.
The multi-epitope peptide may be administered in a simple manner using an administration route including injection (subcutaneous or intravenous), rhinenchysis, instillation, oral administration, inhalation, percutaneous administration, etc.
The one-letter notation for amino acids used in the specification and the sequence listing follows the definition prescribed by IUPAC, Commission on Biochemical Nomenclature (cf., Biochemical Dictionary, 2nd ed., 1468, Table 1.1).